Isolation of cellular material under microscopic visualization

ABSTRACT

A method of microdissection which involves: forming an image field of cells of the tissue sample utilizing a microscope, identifying at least one zone of cells of interest from the image field of cells which at least one zone of cells of interest includes different types of cells than adjacent zones of cells, and extracting the at least one zone of cells of interest from the tissue sample. The extraction is achieved by contacting the tissue sample with a transfer surface that can be selectively activated so that regions thereof adhere to the zone of cells of interest to be extracted. The transfer surface includes an activatable adhesive layer which provides chemical or electrostatic adherence to the selected regions of the tissue sample. After the transfer surface is activated the transfer surface and tissue sample are separated. During separation the zone of cells of interest remains adhered to the transfer surface and is thus separated from the tissue sample.

RELATED APPLICATION

The present application is a divisional of application Ser. No.08/925,894, filed Sep. 8, 1997, now U.S. Pat. No. 6,010,888, which is adivisional of Ser. No. 08/544,388, filed Oct. 10, 1995, now U.S. Pat.No. 5,843,657, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/203,780, filed Mar. 1, 1994 now U.S. Pat. No.5,843,644.

TECHNICAL FIELD

The present invention relates to methods and devices for the analysis ofcellular samples. More particularly, the present invention relates tomethods and devices for the microdissection and analysis of cellularsamples which may be used in combination with a number of differenttechnologies that allow for analysis of enzymes, MRNA and DNA from purepopulations or subpopulations of particular cell types.

BACKGROUND ART

Many diseases are now understood at the molecular and genetic level.Analysis of such molecules is important for disease diagnosis andprognosis. Previous methods for direct extraction of cellular tissuematerial from a tissue sample are limited because the extractionreflects only the average content of disease associated markers. Inreality, tissues are very heterogeneous, and the most diagnosticportions of the tissue may be confined to a few hundred cells or less ina lesion.

Normal tissue samples contain a variety of cell types surrounding andadjacent to the pre-invasive and invasive tumor cells. A region of thetumor tissue subject to biopsy and diagnosis as small as 1.0 mm cancontain normal epithelium, pre-invasive stages of carcinoma, in-situcarcinoma, invasive carcinoma, and inflammatory areas. Consequently,routine scraping and cutting methods will gather all of these types ofcells, and hence, loss of an allele will be masked by presence of anormal copy of the allele in the contaminating non-malignant cells.Existing methods for cutting away or masking a portion of tissue do nothave the needed resolution. Hence the analysis of genetic results bythose previous are always plagued by contaminating alleles from normalcells, undesired cells or vascular cells.

The molecular study of human tumors is currently limited by thetechniques and model systems available for their characterization.Studies to quantitatively or qualitatively asses proteins or nucleicacid expression in human tumor cells are compromised by the diverse cellpopulations present in bulk tumor specimens. Histologic fields ofinvasive tumor typically show a number of cell types including tumorcells, stromal cells, endothelial cells, normal epithelial cells andinflammatory cells. Since the tumor cells are often a relatively smallpercentage of the total cell population it is difficult to interpret thesignificance of net protein or nucleic acid alterations in thesespecimens.

The processes of tumor invasion and metastasis depend upon increasedproteolytic activity of invading tumor cells. Matrix metalloproteinases,cathepsins B, D, and L, and plasminogen activator have been implicatedin the metastatic cascade. Cathepsin D has been suggested to be anindependent marker of prognosis in breast cancer. Several lines ofcorrelation evidence support the concept that proteases are important intumor invasion including: increased protease activity and/or alteredsubcellular distribution of proteases in highly metastatic tumor celllines, increased protease expression in invasive human tumors asdetermined by both immunohistochemistry and assays of tumor tissuehomogenates, and increased MRNA levels in human tumors. All of thesetechniques have generated important information regarding proteaseexpression in human tumors, however, they have not provided definitiveevidence that proteases are up-regulated in specific Legions where tumorinvasion is occurring.

Studies of human tumor cells in culture do not account for the complexinteractions of the tumor cells with host cells and extracellularmatrix, and how they may regulate tumor cell protease productivity or isactivation. Immunohistochemical staining allows one to examine enzymedistribution in regions of tumor invasion, however, results vary withtissue fixation and antibody-antigen affinity, and provide only asemi-quantitative assessment of protein levels. Furthermore,quantitative interpretation of staining results is complicated by thevariability of staining patterns within tissue sections, subjectiveevaluation of staining intensity, and the difficulty in interpreting thesignificance of stromal staining. In addition, many antibodies utilizedin the study of proteases do not differentiate pro-enzyme from activeenzyme species. Assays of enzyme or MRNA levels from homogenates ofhuman tumors does not account for either the mixed population of cellswithin the specimens, or the concomitant pathophysiologic processeswhich may be occur in the tissue.

Human tumors accumulate genetic abnormalities as they develop from asingle transformed cell to invasive and metastatic carcinoma.Identification and characterization of the genes which are mutated, lostor abnormally regulated can provide important insights for cancerdiagnosis, prognosis, and therapy. Furthermore, identification of suchgenetic lesions may facilitate early diagnosis by definitiveidentification of premalignant lesions so they can be treated beforethey progress to invasive cancer.

A general dictum of cancer progression states that cells can betransformed after acquiring two separate alterations in the tumorsuppressor gene. Subsequent tumors progress stepwise from dysplasticlesions to in-situ, to invasive and metastatic neoplasms. In-situcarcinomas are frequently observed arising in association with aspectrum of epithelial hyperplasias and larger invasive tumors are oftenassociated with regions of carcinoma in-situ at the tumor periphery.

Pathologists have historically interpreted a side-by-side association ofatypical hyperplasia, in-situ carcinoma, and invasive tumors as evidenceof a cause and effect relationship among the entities. However, littleindirect evidence existed previously which supports this model.

Prior methods of study have not allowed investigators to specificallyexamine genetic alterations in pre-invasive lesions. The presentinvention provides a novel improved means to specifically examinegenetic alterations in pre-invasive lesions of common epithelial tumorssuch as breast and prostate carcinoma. In particular the presentinvention permits the microsampling of five or less cells with RNA andDNA extraction of the sampled cells. This method has been demonstratedto be extremely sensitive and to surpass previous and currenttechnologies by more than two orders of magnitude. It has allowed thesensitive detection of loss of heterozygosity in early pre-invasivelesions being a gateway to the discovery of a new genetic loci onchromosome 11 for breast cancer and a new genetic loci on chromosome 8for prostate carcinoma.

DISCLOSURE OF THE INVENTION

It is according one object of the present invention to provide a methodof identifying specific cells in cellular tissue sample.

Another object of the present invention is to provide a method of directextraction of specific cells from a cellular tissue sample.

It is a further object of the present invention to provide an automatedmethod of identifying specific cells in cellular tissue sample.

A further object of the present invention is to provide an automatedmethod of direct extraction of specific cells from a cellular tissuesample.

A still further object of the present invention is to provide a methodof obtaining pure cell populations from a cellular tissue samples.

According to these and further objects of the present invention whichwill become apparent as the drescription thereof proceeds, the presentinvention provides for a method of direct extraction of cellularmaterial from a tissue sample which involves:

providing a tissue sample;

contacting the tissue sample with a transfer surface which can beactivated to provide selective regions thereof with adhesivecharacteristics;

identifying at least one portion of the tissue sample which is to beextracted;

activating a region of the transfer surface which is in contact with theat least one portion of the tissue sample so that the activated regionof the transfer surface adheres to the at least one portion of thetissue sample; and

separating the transfer surface from the tissue sample while maintainingadhesion between the activated region of the transfer surface and the atleast one portion of the tissue sample so that the at least one portionof the tissue sample is extracted from a remaining portion of the tissuesample.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will be described with reference to the attacheddrawings which are given by way of non-limiting examples only, in which:

FIG. 1 is a functional system diagram which shows how a tissue sample ismicroscopically imaged, displayed on a display monitor, and how a regionof the imaged sample is selected and identified for subsequentmicrodissection and analysis.

FIGS. 2a-2 c are a series of functional system diagrams which show how azone of tissue sample is extracted from the slide-mounted tissue sampleaccording to one embodiment of the present invention.

FIG. 3 is a schematic illustration of an alternative device forextracting sample zones from the 9:slide-mounted tissue sample.

FIGS. 4a and 4 b are schematic diagrams of a manual extraction toolmanipulator which can be used together with the extraction device ofFIG. 3 according to the present invention.

FIG. 5 is a functional system diagram which shows how a zone of sampletissue can be directed to an appropriate analysis protocol.

FIG. 6a and 6 b show the expression of MMP-2 in ten invasive coloncarcinoma cases (FIG. 6a) and in five cases of invasive breast carcinoma(FIG. 6b) as compared to normal colonic mucosa from the same patients.

FIG. 7 shows SSCP analysis of MMP-2 activation site.

FIGS. 8a-8 d are schematic illustrations of the sequential steps of anadhesive transfer method according to one embodiment of the presentinvention.

FIG. 9 shows the results of a sequencing gel electrophoresis of PCRamplified DNA from human tissue microdissected by the method depicted inFIGS. 8a-8 b.

BEST MADE FOR CARRYING OUT THE INVENTION

The present invention is directed to a method of analyzing cellularmaterial on a molecular or genetic level which involves: visualizing afield of cells in a tissue sample under a microscope, contacting anidentified area with a surface which simultaneously dissolves, extractsand/or retains a cellular material of interest, and transferring thecellular material of interest to a suitable analysis system. The presentinvention is particularly applicable to the analysis of local tissueenzymes, antigens, DNA, RNA, and the like.

According to a preferred embodiment, the present invention is directedto adhesive transfer methods which involve microscopic visualization andtransfer of cellular material to a procurement or transfer surface.

The present invention is also directed to a fully automated systemwhereby a tissue can be visualized on a screen, so that a precise fieldof cells of interest can be identified by a variety of labels,circumscribed, and then be automatically extracted and analyzed.

FIG. 1 is a functional system diagram which shows how a tissue sample ismicroscopically imaged, displayed on a display monitor, and how a regionof the imaged sample is selected and identified for subsequentmicrodissection and analysis. As depicted in FIG. 1, a tissue sample 1is provided on a glass slide 2 for microscopic examination and imaging.The sample tissue 1 can be fixed on the glass slide 2 according to anyconventional method, including attachment to the glass slide 2 with anagarose gel, fixing the tissue sample in paraffin, etc.

The glass slide 2 having the sample tissue 1 mounted thereon is placedon the stage of a microscope. The microscope, generally indicated byreference numeral 3 receives an image of the tissue sample 1. A videocamera (not shown) is connected to the microscope 3. The video camerareceives the image of the sample tissue 1 from the microscope 3 anddisplays the image of the tissue sample on a display monitor 4.

The image of the sample tissue 1 is limited to the “field” of themicroscope 3 for any given image. As indicated in FIG. 1, the field ofthe sample tissue image may include several zones, “A”, “B”, “C”, and“D” of different types of cells which can be optically distinguished byutilizing a suitable dye(s) to stain the tissue sample. For exemplarypurposes, FIGS. 1 and 2a-2 c assume that zone “B” is the zone ofcellular material of interest. The image on the display monitor 4 isused by the operator to select and identify one or more zones of thetissue sample 1 which are of interest. According to one embodiment ofthe present invention, after the zone(s) of interest are selected andidentified, the operator manually manipulates a device to extract theidentified zone(s) from the glass slide 2. The extracted zone(s) ofsample material may either include an analysis sample. Otherwise, theidentified and extracted zone(s) can include zones which are todiscarded and the remaining zone(s) which are retained on the glassslide 2, can be later analyzed.

In addition to manual operation which is discussed in more detail below,it is possible, according to another embodiment of the presentinvention, to utilize the image on the display monitor 4 to select andidentify a sample zone(s) whose relative position is determinedutilizing a computer which receives a digitized signal of the image fromthe video camera (or microscope), and which receives a referenceposition of the stage of the microscope 3 upon which the sample is held.Such positioning detection and recognition systems are conventional inthe art and can be readily applied to automate the sample preparationmethod of the present invention. In this automated embodiment of theinvention, the computer which performs the positioning detection andrecognizing can also be used to control the movement of the devicesdiscussed below that are used to extract tissue zones, thus automatingthe sample removal. In addition, the image of the sample can beelectronically scanned to automatically identify zones having apredetermined or relevant degree of staining, musing known techniquesand devices. Thus, in a preferred embodiment, a computer could be usedto select and identify zones of interest and the relative position ofsuch zones, for manipulating a device to remove such zones in anautomated manner.

FIGS. 2a-2 c are a series of functional system diagrams which show how azone of tissue sample 1 is extracted from the slide-mounted tissuesample 1 according to one embodiment of the present invention. It is tobe understood that the steps depicted in FIGS. 2a-2 c could be eitherpreformed manually by an operator or by a computer utilizingconventional positioning and control methods, e.g. computer controlledrobotics.

The embodiment of the invention depicted in FIGS. 2a-2 c utilize acontact probe 5 which has an adhesive/extraction reagent 6 on the tipthereof. A suitable adhesive/extraction reagent can include a mixture ofpiccolyte and xylene. In FIG. 2a the contact probe 5 is positionedeither manually or by computer control so as to be above and alignedwith the sample zone (“B”) to be extracted. As can be readily understoodfrom FIG. 2a, the surface area of the contact probe tip (andadhesive/extraction reagent) needs to be about equal to, and no greaterthan, the surface area of the zone to be extracted. Otherwise, excessiveremoval of adjacent tissue zones will occur.

Once the tip of the contact probe 5 is aligned with the sample zone(“B”) to be extracted, the contact probe 5 is lowered so that theadhesive/extraction reagent 6 on the tip thereof contacts the samplezone (FIG. 2b).

The adhesive/extraction reagent 6 is selected to readily adhere to thesample zone. Once the adhesive/extraction reagent 6 on the tip of thecontact probe 5 contacts the sample zone (FIG. 2b) and the sample zonebecomes adhered thereto, the contact probe 5 can be retracted from thecontact position (illustrated in FIG. 2b) and moved as shown in FIG. 2c.Since the relative adhesive force of the adhesive/extraction reagent isgreater than the adhesive force used to mount the sample on the glassslide, the contact probe 5 pulls the sample ozone “B” from the glassslide when withdrawn or retracted. According to one embodiment of thepresent invention, a glass pipette was used as the contact probe

In this embodiment, the tip of the glass pipette was coated with asolution of piccolyte (568 g/l) and xylene (437.5 g/l) by dipping thetip of the glass pipette in the piccolyte/xylene solution.

In addition to removing the sample zone from the glass slide 2, thecontact probe 5 can be used to transfer the extracted sample zone to ananalysis container 7 as indicated in FIG. 2c or to any other location,such as a waste container, a culture media, etc. In a preferredembodiment, the contract probe 5 is used to transfer the extractedsample zone to the sample receiving stage of an automated clinicalanalyzer which is designed to preform a desired analysis of the samplezone. It thus, can be understand that the present invention can providea fully automated method and system for identifying sample zones on aslide-mounted sample, removing sample zones of interest from theslide-mounted sample, and transporting the extracted sample zones to anautomated analyzer which can perform automated analysis of the extractedsample zones.

In FIG. 2c the extracted sample zone is depicted as being dispensed in acontainer 7 which, for example, can be a test tube or similar containerin which analysis on the extracted sample zone can be initiated orperformed. As depicted in FIG. 2c, a reagent solution 8 which removesall or a desired component of the extracted sample zone from the contactprobe tip can be placed in the container 7 before the extracted samplezone is deposited therein. For example, in the case of DNA analysis, asolution of Tris (50 mM, pHB.5), EDTA (1 mM), Tween 20 (0.5%), andproteinase K (0.2 mg/mL) can be used to remove the extracted sample zonefrom the tip of the contact probe 5 and dissolve the tissue material foranalysis purposes.

In addition to the contact probe depicted in FIGS. 2a-2 c, a hollowsuction probe could also be used to extract sample zones from theslide-mounted tissue sample 1. Such a suction probe could be providedwith sharp annular tip by which sample zones could be punched out andextracted by suction forces.

FIG. 3 is a schematic illustration of an alternative device forextracting sample zones from the slide-mounted tissue sample 1. Theextraction device 9 shown in FIG. 3 includes a cutting blade 10 and agrasping arm 11. The grasping arm 11 can be moved in an opposed mannerwith respect to the cutting blade 10. The grasping arm 11 is shown inits open position in FIG. 3. The grasping arm 11 is movable between theillustrated open position to a closed position in which the tip of thegrasping arm 11 contacts the cutting blade 10. The movement of thegrasping arm 11 can be controlled by a cable and pulley system in whichgrasping arm 11 is caused to pivot at its base by applying tension to acable which passes through a pulley located at the base of the graspingarm 11. The tension on the cable can be applied by actuating a lever ordepressing a button 12 on the device which applied tension to the cablein a known manner. Such actuating mechanical structures are known in theart of gripping devices.

In operating the device of FIG. 3, the cutting blade 10, which is at anobtuse with respect to the central axis of the device can cut out andscoop up a portion of a tissue sample by placing the cutting blade 10 onone edge of a portion of the tissue sample to be extracted and thenmoving the grasping arm 11 into the closed position. As the grasping arm11 comes into contact with the tissue sample, it draws the cutting blade10 into the sample and presses a portion of the sample toward thecutting blade 10 thereby causing a portion of the sample contactedbetween the cutting blade 10 and the grasping arm 11 to be cut out andscooped up from the sample.

In a further, alternative embodiment of the device of FIG. 3, themovement of the grasping arm 11 can be effected by a toothed gearinstead of a pulley and a cooperating toothed rod in place of a cable.Such mechanical structures are known in the art of gripping devices.

FIGS. 4a and 4 b are schematic diagrams of a manual extraction toolmanipulator which can be used together with the extraction device ofFIG. 3 according to the present invention. In FIG. 4a the extractiontool manipulator is depicted as having a base 13 equipped with aclamping means 14 for removable attaching the device to a brace orsupport portion of the stage of a microscope (see FIG. 4b). The clampingmechanism includes a clamping plate 15 that is secured to a threadedshaft 16 which passes through a threaded bore 17 in a lower portion ofthe base 13. A tightening knob 18 is provided on the end of the threadedshaft 16. Turning the tightening knob 18 causes the clamping plate 15 tomove with respect to an upper portion 19 of the base 13. Thus, theextraction tool manipulator can be clamped to a portion of the stage ofa microscope 20 as depicted in FIG. 4b by positioning a brace or supportportion 21 of the stage of the microscope 20 between the clamping plate15 and the upper portion 19 of the base 13 and turning knob 18 totighten the clamping plate 15 against the brace or support portion 21 ofthe stage of the microscope 20.

The extraction tool manipulator includes a tool holder 22 having athrough-bore 23 therein for receiving the shaft of an extraction tool24. Ideally, the tool holder 22 should allow for damped fore and aftmovement of the extraction tool. Therefore, according to a preferredembodiment, the through-bore 23 of the tool holder 22 contains a bushingwhich can be adjustable tightened against the tool shaft by tool lockingscrew 24.

The tool holder 22 is supported by support shaft 25 which is connectedat opposite ends by 360° damped swivels 26 and 27 to the tool holder 22and the base 13. The length of the support shaft 25 between the 360°damped swivels 26 and 27 is adjustable. The adjustment of theindependent 360° damped swivels 26 and 27 together with the adjustablelength of the support shaft 25 and the position of the tool shaft withinthrough-bore 23, allows a high degree of movement of the extraction toolwith respect to a slide-mounted sample positioned on the stage of themicroscope. Therefore, an operator can manipulate an extraction toolheld by the extraction tool manipulator and remove selected tissue zonesfrom a slide-mounted tissue sample with a high degree of precision.

FIG. 5 is a functional system diagram which shows how a zone of sampletissue can be directed to an appropriate analysis protocol. As depictedin FIG. 5 a microextraction of a zone of tissue sample can be taken froma slide-mounted tissue sample 1 as discussed above and transferred to asample preparation stage 28 in which the cells of interest can beextracted and collected for analysis. Excised cells may also besolubilized at this stage. If the cells of interest contain DNA or RNAthe extracted sample is subjected to polymerase chain reaction (PCR)amplification and hybridization, strand conformational polymorphism, andsouthern and northern blotting as desired. If the cells of interestcontain proteins, the extracted sample can be subjected to enzymezymography, an immunoassay, or a biochemical assay.

Selective extraction or microdissection of frozen tissue sectionsaccording to the present invention allows for recovery and analysis ofboth active enzymes and MRNA. Additionally, the DNA recovered from thesesections is in the native condition and can be used for studies such asDNA fingerprinting. Microdissection of paraffin embedded tissuesaccording to the present invention allows for PCR amplification of DNAfrom pure cell populations representing less than one high poweredfield, or a single layer of epithelial cells lining cystic spaces.

For general preparation of samples for frozen section microdissectionaccording to the present invention microdissection slides can beprepared by placing 1% agarose on a standard histology slide and coverslipping. After a short period of time, e.g., 5 minutes the cover slipis removed leaving a thin gel on the slide. A small frozen tissuesection, e.g. 25 micron thick, is placed on the agarose gel and brieflystained with eosin. The tissue may also be treated with agents todenature RNase depending on the subsequent extraction method. Underdirect microscopic visualization the specific cell population orsub-population of interest is procured from the tissue section utilizingthe techniques discussed above.

For enzyme analysis the procured tissue specimen can be placed in anappropriate buffer depending on the enzyme of interest. The enzymelevels can be measured by several methods including zymography and theuse of specific fluorometric substrates. The precise levels of enzymeexpression in a specific cell population can be determined.

For messenger RNA analysis the tissue specimen can be placed on agaroseand treated with agents to denature RNase if necessary. The procuredtissue specimen is immediately frozen in liquid nitrogen. The tissue canbe used immediately or stored at −70° C. for several months. The MRNAcan be extracted using an oligo dT column (Micro-Fast track MRNAIsolation Kit, Invetrogen Co.). The recovered MRNA of the pure cellpopulations can be amplified and investigated using PCR technology.

For DNA analysis the tissue specimen can be placed in a single stepextraction buffer solution of 50 mM Tris, pH 8.5; 1 mM EDTA, 0.5% Tween20, and 0.2 mg/ml proteinase K, incubated for four hours at 37° C.,followed by ten minutes incubation at 95° C. The recovered DNA can beamplified and analyzed by PCR technology. If native DNA is required forDNA fingerprinting, the proteinase K can be added after DNase.

For paraffin section microdissection routine formalin fixed, paraffinembedded tissue sections are microdissected after deparaffinization andbrief staining with eosin. Tissue sections are visualized by directmicroscopy and cell populations or subpopulations of interest areprocured using a modified glass pipette with the adhesive coated tipdiscussed above. Tissue specimens as small as one cell can be procuredwith this method. The specificity of dissection represents a significantimprovement over currently known techniques.

For DNA analysis of paraffin embedded tissue, the glass pipette with thedissected tissue specimen is placed in a single step extraction buffersolution of 50 mM Tris, pH 8.5; 1 mM EDTA, 0.5% Tween 20, and 0.2 mg/mlproteinase K which removes the tissue from the pipette tip. Depending onthe size of the sample it is incubated from two to twenty-four hours at37° C., followed by a ten minute incubation at 95° C. The glass pipettetip can then be sterilized and reused.

Features and characteristics of the present invention will beillustrated by the following examples to which the present invention isnot to be considered limited. in the examples and throughout percentagesare by weight unless otherwise indicated.

The following examples were performed in an attempt to establish if thepresent invention could be used to A more specifically study proteasedistribution during human tumor invasion. Levels of MMP-2 and cathepsinB in fields of invasive breast and colon carcinoma were measured toassess if the enzymes in these regions were quantitatively increased ascompared to matched numbers of normal cells from the same patient.

In the following examples, normal and tumor samples of colon and breasttissue from surgical resections were maintained in a frozen condition(−70° C.) until analysis. Tissue section of invasive breast and coloncarcinoma were selected based upon histologic evaluation. For the tumorsections histologic fields of tissue which contained invasive tumor andstroma were selected, but not normal epithelium or significant numbersof inflammatory cells. The control sections of normal tissue containedepithelium and a thin section of underlying stroma. The proportion ofepithelial and stromal tissue was similar for both normal and tumorsections.

In the examples microdissection slides were prepared by coveringstandard histology slides with 200 microliters of warm agarose (1%) andover laying a cover slip. After five minutes the coverslip was removedleaving a thin bed of agarose on the slide. Twenty micron thick frozensections were prepared in a cryostat and placed on the agarose gel. Thetissue was briefly dipped in eosin. Optimum microdissection was achievedby starting at the edge of each section and systematically dissectingand separating histologic fields of interest with the microdissectingdevice of FIG. 3. Areas of interest were retained on the slide forsubsequent reanalysis. The DNA content of the specimens was determinedby spectrophotometric measurement at 260 nm. The DNA content of eachsample was proportional to the number of cells counted in eachhistologic section.

EXAMPLE 1

In this example, samples of normal and tumor tissue matched for cellnumber were analyzed from each subject. Levels of MMP-2 were determinedby zymography and quantified using an Arcus scanner. Results werestatistically analyzed using the students t-test. Cathepsin B levelswere determined as V_(max) against the substrate Z—Arg—Arg—NHMec.

The results of this example are set forth in Table 1 below which liststhe cathepsin B activity in matched pairs of invasive coloncarcinoma/normal epithelium, and invasive breast carcinoma/normalepithelium. Activity measurement are expressed as V_(max), nmol/min×mgDNA. Cathepsin B activity was increased an average of 2.3 fold in thecolon tumors (p<0.005), and 6.9 fold in the breast tumors (p=0.077).

TABLE 1 SAMPLE NORMAL TUMOR TUMOR/NORMAL CATHEPSIN B ACTIVITY ININVASIVE HUMAN COLON CARCINOMA 1 1.38 4.75 3.4 2 1.89 2.25 1.2 3 1.986.32 3.2 4 0.49 1.88 3.8 5 0.44 0.72 1.6 6 1.03 1.92 1.9 7 0.47 1.35 2.98 0.19 0.33 1.7 9 1.07 0.90 0.8 10  0.33 0.88 2.7 Average 0.93 2.13 2.3CATHEPSIN B ACTIVITY IN INVASIVE HUMAN BREAST CARCINOMA 1 0.63 3.02 4.82 0.51 10.08 19.8 3 0.61 4.43 7.3 4 2.21 2.38 1.1 5 2.06 3.72 1.8Average 1.20 4.73 6.9

As can be seen from Table 1, all five breast tumors and nine of the tencolon tumors showed increased activity of cathepsin B as compared tomatched numbers of normal cells from the same patient (Table 1).Increased activity in the colon tumors ranged from 19% to 283%, with anaverage increase in tumors of greater than two fold. The increase ofcathepsin B activity was more pronounced in breast tumors with anaverage increase of slightly less than seven fold.

EXAMPLE 2

In this example, polymerase chain reaction (PCR) analysis was preformed.On the basis of previously reported cDNA sequences of 72 kDa type IVcollagenase, sense and antisense oligonucleotide primers weresynthesized for amplification of the enzyme activation site (M. Onistoet al, “Reverse Transcription-Polymerase Chain Reaction Phenotyping ofMetalloproteinases and Inhibitors in Tumor Matrix Invasion”, Diagn. Mol.Pathol, 2(2):74-80, 1993). The paired oligonucleotide sequences were:5′-CAA TAC CTG AAC ACC TTC TA, 3′-CTG TAT GTG ATC TGG TTC TTG. LabeledPCR for Single Strand Conformation Polymorphism (SSCP) was obtained bycombining the following in a 10 microliter reaction: 1 microliter 10×PCR buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl₂; 0.1% w/vgelatin); 1 microliter of EDNA extraction buffer; 50 pmol of eachprimer; 20 nmol Beach of dCTP, dGTP, dTTT, and DATP; 0.2 microliter[³²P]dCTP (6000 Ci/mmol); and 0.1 unit Taq DNA polymerase. Theamplification reaction was carried out for 30 cycles at 95° C. for 30 s,60° C. for 30 s, and 72° C. for 30 s.

FIG. 5a shows the expression of MMP-2 in ten invasive colon carcinomacases as compared to normal colonic mucosa from the same patients. Thebar graphs show increases of approximately three fold in the 72 kDapro-form of the enzyme (p<0.001) and ten fold in the 62 kDa active formof the enzyme (p<0.001).

FIG. 6b shows the expression of MMP-2 in five cases of invasive breastcarcinoma. The bar graphs show an appropriate increase of three fold inthe 72 kDa pro-form of the enzyme (p<0.05) and ten fold in the 62 kDaactive form of the enzyme (p<0.05).

The 72 kDa pro-type IV collagenase and 62 kDa active form of the enzymewere increased in all ten colon tumors and all five breast tumors ascompared to normal tissue from the same patient. The increase wasgreater in the 62 kDa active form of the enzyme which was elevated anaverage of ten-fold in both the colon and breast tumors as compared tonormal control tissue. The 72 kDa pro-enzyme levels were increased anaverage of three fold in both tumor types. For both breast and colontumors the increase in the 62 kDa active enzyme was more variable thanthat of the pro-enzyme. Elevations in the 62 kDa active enzyme in tumorsranged from 3 to 20 fold while increases in the 72 kDa pro-enzyme wereconsistently in the 2 to 5 fold range. These results are similar to therecent findings of Davis et al (“Activity of Type IV Collagenases inBenign and Malignant Breast Disease”, Br. J. Cancer, 67:1126-1131, 1993)in their analysis of human breast tumors. These authors performedzymogram analysis of tissue sections from human breast cancer patients.These analyses demonstrated that the fraction of total MMP-2 present asthe 62 kDa activated form was statistically elevated in malignantdisease, and a high proportion of this active enzyme species wasdetected in higher grade tumors. The present invention extends thisanalysis by comparing and quantitating both 72 kDa and 62 kDa forms ofthe enzyme in specific regions of invasive tumor and matched normalcontrol epithelium from the same patient.

EXAMPLE 3

In this example, strand conformation polymorphism (SSCP) analysis waspreformed. Labeled amplified DNA was mixed with an equal volume offormamide loading dye (95% formamide; 20 mM EDTA; 0.05% bromophenolblue, and 0.05% xylene cyanol). The samples were denatured for 5 min at95° C. and loaded onto a gel consisting of 6% acrylamide (49:1acrylamide:bis), 5% glycerol, and 0.6× TBE. Samples were electrophoresedat 8 W at room temperature overnight. Gels were transferred to 3 mmWhatman paper, dried and autoradiography was performed with Kodak X-OMATfilm.

FIG. 7 shows SSCP analysis of MMP-2 activation site. The figure showsrepresentative cases of normal colon is mucosa compared to invasivecolon carcinoma, and normal breast tissue compared to invasive breastcarcinoma. No difference is observed between the normal and tumorspecimens. The two band in each lane represent single and double formsof DNA. Similar results were obtained for ten colon carcinomas and fourbreast carcinomas.

To assess if increased tumor levels of activated MMP-2 are due to amutation in the enzyme, PCR was used to amplify DNA sequence coding forthe activation site of gelatinase A from the colon and breast tumors.The activation site is located 10 kDa from the N-terminus of the enzymeand contains the site of cleavage which converts the 72 kDa pro-enzymeinto the 62 kDa active species. Amplification and analysis of thisregion by PCR and SSCP showed no detectable mutations in any of the tencolon tumors or four breast tumors studied. These results suggest thatincreased levels of active enzyme in invasive tumors is most likely dueto a tumor associated activating species. The sensitivity of PCRamplification of DNA from microdissected frozen tissue sections wasdetermined to be less that one high power field. Similar to theamplification of DNA, amplification of mRNA from small cell populationswas preformed according to the present invention using reverse PCR.

A previous study indicated that MMP-2 is up-regulated in human coloncarcinoma. However, recently several studies using in situ hybridizationanalysis report that the MRNA level of MMP-2 in human colon carcinoma isincreased in the stromal cells as opposed to the tumor cells. In orderto address this possibility frozen tissue sections were microdissectedto measure enzyme levels of MMP-2 in separate tumor and stromal cellpopulations. From a single high power field sufficient tissue wasrecovered to quantitate enzyme levels by zymography. Studies of invasivetumor cells and adjacent stroma from three cases indicate that 72 kDapro-MMP-2 and active 62 kDa form are associated with both tumor cell andstromal cell populations. Preliminary data suggest that the highestenzyme levels are at the tumor-stromal interface.

According to a preferred embodiment, the present invention is directedto adhesive transfer methods which involve microscopic visualization andtransfer of cellular material to a procurement or transfer surface.

According to the general procedure, an adhesive surface is placed incontact with the surface of the cells or tissue and the adhesive forcebinds the cellular material of interest to the adhesive surface. Theadhesive surface which can be the tip of a tool or needle is used toprocure the material and transfer it to a liquid analysis reactionmixture. Examples of adhesive surfaces include adhesive coatings on thetip of the tool, or the use of electrostatic forces between the tip andthe surface of the cellular material.

As described in detail below, the isolation and transfer methods of thepresent invention can involve a specialized continuous activatableadhesive layer or surface which is applied to the cellular material overan area larger than the area selected for microscopic procurement. Theadhesive function of the subsection of the surface in contact with thearea selected for procurement is activated by electromagnetic orradiation means. According to a preferred embodiment a laser or otherelectromagnetic radiation source is used to activate the adhesive forcesbetween the cellular material and the activatable adhesive layer orsurface. This allows for accurate generation of adhesive forces only inthe precise microscopic area selected. Suitable lasers includediode-pumped Nd, YAG and Nd:Yag, tunable single frequency Ti:sapphirelasers, solid state lasers. Lasers having wavelength outputs fromultraviolet to infrared can be used according to the present invention.

In addition to lasers, it is possible to activate adhesive layerutilizing electrically heated radiation heaters or heated probes,focused or masked non-laser light sources such as flashbulbs, xenonlamps, etc.

FIGS. 8a-8 d are schematic illustrations of the sequential steps of anadhesive transfer method according to one embodiment of the presentinvention.

As depicted in FIG. 8a, the adhesive transfer method utilizes a transfersurface 30 which includes a backing layer 31 and an activatable adhesivelayer 32. In procedures which utilize laser activation of the adhesivelayer, the backing layer 31 is preferably transparent, e.g. made of atransparent polymer, glass, or similar material. The activatableadhesive layer 32 can be an emulsion layer, a coated film, or a separateimpregnated web fixed to the backing layer. Examples of materials fromwhich the adhesive layer 32 can be make include thermal sensitiveadhesives and waxes (e.g., #HAL-2 180C from Precision Coatings), hotglues and sealants (available from Bay Fastening Systems, Brooklyn,N.Y.), ultraviolet sensitive or curing optical adhesives (e.g.,N060-N0A81, ThorLabs Inc.), and thermal or optical emulsions (e.g.,silkscreen coated emulsion B6 Hi Mesh, Riso Kagaku Corp.) The backinglayer 31 provides physical support for the adhesive surface, and thuscan be integrated physically into the activatable adhesive surface.

The activatable adhesive layer 32 is characterized by its ability to bestimulated (activated) by electromagnetic radiation so as to becomelocally adherent to the tissue. For purposes of selectively activatingthe activatable adhesive layer 32 one or more chemical components canincorporated into the layer, which chemical components cause selectiveabsorbance of electromagnetic energy.

As depicted in FIG. 8a, the transfer surface 30 is initially positionedover a cellular material sample 33 which can be a microtome section orcell smear which is supported on a support member 34 which can be amicroscopic slide. In the case of a tissue microtome, routine procedurescan be used to provide paraffin embedded, formalin-fixed tissue samples.

As shown in FIG. 8b, the transfer surface 30 is brought into contactwith the cellular material sample 33. It is noted that the activatableadhesive layer 32 preferably has a larger area than the subregion ofcellular material sample which is subsequently selected for procurement.

The transfer surface 30 can be fixed to the cellular material samplesupport 33 by clips, guides, tape, standard adhesives, or similarconvenient means. The transfer surface 30 can also contain a labelregion 35 (see phantom lines in FIG. 8b) to write information such asthe patient's identification code or a test designation.

After the transfer surface 30 is brought into contact with the cellularmaterial sample 33, the cellular material sample is viewed by standardlow or high power microscopy to locate the region of interest “A”. Thisregion can range in size to an area smaller than a single cell (lessthan 10 microns), to a few cells, to a whole field of cells or tissue.When the area of interest “A” is identified, the precise region of theactivatable adhesive layer 32 which is immediately above region “A” isactivated by a beam of electromagnetic energy 36, e.g. a laser beam, sadepicted in FIG. 8c.

Application of the electromagnetic energy 36 causes the region of theactivatable adhesive layer 32 which is immediately above region “A” toadhere to region “A”. Although FIGS. 8c and 8 d depict a single regionof interest “A”, it is to be understood that multiple, discontinuousregions of interest could be selected and procured by appropriate aimingand application of the electromagnetic energy.

As depicted in FIG. 8d, after one or more regions of interest areidentified and the corresponding region(s) of the activatable adhesivelayer 32 is activated by a beam of electromagnetic energy 36, thetransfer surface 30 is detached from the cellular material samplesupport 34. As shown, the removed transfer surface 30 carries with itonly the precise cellular material from the region of interest “A”,which is pulled away from the remaining cellular material sample

As mentioned above, a single transfer surface can be used to remove aplurality of areas of interest from a single cellular material sample.The transfer surface 30 carrying the procured cellular material can betreated with suitable reagents to analyze the constituents of thetransferred material. This can be accomplished by submerging thetransfer surface 30, to which the procured cellular material is adhered,in a suitable reagent solution. Alternatively, one or more of theprocured cellular material regions can be removed from the transfersurface 30, or portions of the transfer surface 30 to which the procuredcellular material are adhered can be punched out of the transfer surface30 and analyzed separately.

In the following Examples 4 and 5, the following sample procurementmethod was followed. 5-10 micron sections of formalin-fixed,paraffin-embedded tissue or froze tissue were prepared on a glass slideaccording to conventional surgical pathology protocol. The paraffinsections were deparaffinized with xylene (×2), 95% ethanol (×2), 50%ethanol (×2), distilled water (×2), and air dried. Frozen or paraffinsections were stained briefly in eosin (1% eosin in 80% ethanol) and airdried.

An adjacent hematoxylin and eosin section was used to assess the tissuesection for optimal areas of microdissection, i.e., localization ofspecific small cell populations of interest, exclusion of regions whichcontain significant inflammation, etc.

Microdissection of selected populations of cells was performed underdirect light microscope visualization. A sterile 30 gage needle was usedas the transfer surface. Electrostatic interaction between the needleand cellular material provided the adherence needed to remove selectedpopulations of cells. It was determined that pure cell populations of aslittle as 5 cells could be procured. In addition it was found possibleto procure cells arranged as a single cell layer, i.e., normalepithelium, epithelial lining of cystic lesions, etc.

EXAMPLE 4

Human prostate cancer has been proposed to progress through an in situtumor phase called prostatic intraepithelial neoplasia (PIN) prior toevolving into overtly invasive cancer. PIN lesions are frequently foundin association with prostate carcinoma, and histologically the cells inPION foci have several features similar to those of invasive prostatecancer cells. Previous reports have shown that PIN lesions arefrequently aneuploid. However the precise relationship between PIN andinvasive carcinoma has remained unclear.

In this Example, frozen normal and tumor prostate samples from 100patients treated with transurethral prostatectomy or radial prostatomywere collected. Of these, 30 cases which contained clearly invasivecancer as well as at least one focus of identifiable PIN were selectedfor study during this Example. Fourteen of the set cases contained morethan one focus of PIN. The histopathology of the tumors was variable andincluded well differentiated, moderately differentiated and poorlydifferentiated. PIN lesions were both low and high grade.

Microdissection of selected populations of normal epithelial cells,cells from PIN lesions, and invasive tumor cells from frozen tissuesections was performed under direct light microscopic visualizationutilizing the method discussed above. Specific cells of interest weremicrodissected and procured from unstained 8 μm frozen sections. In eachcase, normal epithelium, PIN cells, and invasive tumor cells from thesame patient were analyzed.

Procured cells were immediately resuspended in a 20 ml solutioncontaining 10 mM Tris-HCL, pH 8.0, 100 mM ethylenediamine tetraaceticacid (EDTA), It Tween 20, 0.1 mg/ml proteinase K, and incubatedovernight at 37° C. The mixture was boiled for 5 minutes to inactivatethe proteinase K and 0.5-2% of this solution was used for polymerasechain reaction (PCR) analysis.

The oligonucleotide primers D8S136, D8S137, and NEFL were used to locatechromosome 8p12-21. Reactions with D8S137 and NEFL were performed in anMJ Research thermal cycler as follows: 2 minutes at 950° C., followed by40 cycles of: 950° C. for 30 seconds, 620° C. for 30 seconds, 720° C.for 30 seconds, followed by a final 2 minute incubation at 720° C.

Reactions with D8S136 were cycled as follows: 2 minutes at 950° C.,followed by 40 cycles of: 950° C. for 30 seconds, 550° C. for 30seconds, 720° C. for 30 seconds, followed by a final 2 minute incubationat 720° C.

PCR was performed in 12.5 ml reactions with 200 mM DNTP, 0.8 mM primers,2 μl of alpha [³²P]dCTP (NEN), and 1 unit of Taq polymerase. Labeledamplified DNA was mixed with an equal volume of formamide loading dye(95% formamide; 20 mM EDTA; 0.05% bromophenol blue, and 0.05 xylenecyanol).

The samples were denatured for 5 min at 950° C. and loaded into a gelconsisting of 7% acrylamide (49:1 acrylamide:bis), 5.7 M urea, 32%formamide, and 0.089 M Tris, 0.089 M borate. 0.002 M EDTA (1× TBE).Samples were electrophoresed at 95 Watts for 2-4 hours. Gels weretransferred to 3 mM Whatman paper, and autoradiography was performedwith Kodak X-OMAT film. The criterion for LOH was complete, or nearcomplete absence of one allele as determined by visualization. Caseswith LOH showed two alleles in the normal epithelium control and oneallele in the tumor or PIN all with similar intensities. Cases withcomplete or near complete loss (i.e., very faint band) of one allele intumor or PIN were considered positive for LOH at that marker.

The present inventive method was used to microdissect cells from tissuesections to study loss of heterozygosity on chromosome 8p12-21 inpatients with both prostatic carcinoma and adjacent foci of PIN. Tissuemicrodissection was conducted on 30 patients with concomitant PIN andinvasive prostate cancer. In each case normal epithelium, invasiveprostate cancer and at least one focus of PIN from the same patient wereexamined. In 14 cases multiple foci of PIN were examined. In all caseseach individual PIN lesion and corresponding invasive tumor wereselectively microdissected from of adjacent stroma, normal epitheliumand inflammatory cells. Essentially pure populations of cells ofinterest were procured.

LOH on chromosome 8p12-21 occurred in at least one PIN lesion in 26 of29 (89.6%) informative cases. Fourteen of the cases contained more thanone PIN lesion. Eleven of these cases showed different allele losspatterns among the PIN lesions, including loss of opposite alleles. Intotal, 8p12-21 LOH was seen in 63.6% (35/55) of PION lesions studied.Allelic loss of chromosome 8p12-21 was seen in invasive tumors in 28 of29 (96.5%) patients. In contrast with the success associated with theadhesive transfer technique of the present invention, the use of ascraping dissection technique produced an LOH of less than 15%. Thisindicates the sensitivity of the adhesive transfer of the presentinvention is much greater than conventional techniques.

EXAMPLE 5

Nascent in situ breast carcinomas are frequently observed arising inassociation with a spectrum of epithelial hyperplasias and invasivecarcinoma. Pathologists have historically interpreted the commonassociation of atypical hyperplasia, in situ carcinoma and invasivecarcinomas as evidence for a relationship among the entities.

The polymorphic DNA marker used in this Examiner was PYGM located onchromosome 11q13. Reactions were cycled in a thermal cycler as follows:94° C. for 1.5 min., 55° C. for 1 min., 72° C. for 1 min. for a total of35 cycles. PCR was performed in 10 μl volumes and contained 1 μl 10× PCRbuffer (100 mM Tris-HC1, pH 8.3; 500 mM KCl; 15 mM MgCl₂; 0.1% w/vgelatin; 2 μl of DNA extraction buffer, 50 pM of each primer; 20 nM eachof dCTP, dGTP, dTTP, and dATP; 0.2 μl [³²P]DCTP (6000 Cl/M); and 0.1unit Taq DNA polymerase. Labeled amplified DNA was mixed with an equalvolume of formamide loading dye (95% formamide; 20 mM EDTA; 0.05%bromophenol blue; and 0.05% xylene cyanol). The samples were denaturedfor 5 min. at 95° C. and loaded into a gel consisting of 6% acrylamide(49:1 acrylamide:bis). Samples were electrophoresed ar 1800 volts for2-4 hours. Gels were transferred to 3 mM Whatman paper, dried andautoradiography was performed with Kodak X-OMAT film. The criterion forLOH from the microdissected in situ and invasive breast samples wascomplete absent of an allele.

Using the adhesive transfer technique of the present invention, cellswere microdissected from normal epithelium, in situ carcinoma andinvasive carcinoma from 8 μm thick formalin fixed deparaffinizedsections from individual biopsies. Allelic loss of chromosome 11q13 wasfound in 69% of human breast carcinoma cases studied (n=105). Theallelic loss was observed in both the in situ and invasive components ofthe tumors. In all cases (26/28) where in situ and invasive cancer waspresent in the same section, the identical allele was lost in the insitu and the invasive carcinoma. This provides molecular support for thelong held hypothesis that in situ breast cancer is a precursor toinvasive cancer.

In order to finely map the LOH locus on chromosome 11q13, Genome centerprovided a series of SSCP probes mapped to the relevant region ofchromosome 11. The initial LOH area was determined to be bracketed bythe proximal marker PYGM, and by the distal marker INT-2. A subset of 20of the 105 cases exhibited LOH of either INT-2 or PYGM, but not both.Using these special cases, a series of intervening markers were used tomap the smallest overlapping region between INT-2 and PYGM which showsLOH. It has been possible to pinpoint the LOH zone to a regionencompassed by only one or two YAG or Cosmid clones at a location whichoverlaps with the MEN-1 (Multiple Endocrine Neoplasia type 1) locus.

FIG. 9 shows the results of a sequencing gel electrophoresis of PCRamplified DNA from human tissue microdissected by the method depicted inFIGS. 8a-8 d. Each numbered lane in FIG. 9 is from an individualdissection. Electrostatic transfer is shown in lanes 17-20. Lanes 18-20show complete loss (positive LOH) of the top allele compared to themicrodissected control DNA in lane 17. Thermal infrared transfer(conducted as shown in FIGS. 8c and 8 d) is shown in lanes 22-25. Lane22 (control) shows no DNA transfer without activation. Lanes 23 and 24show complete loss (positive LOH) of top allele group compared tocontrol DNA lane 25.

The above results indicate that microdissection of frozen tissuesections allows for more specific analysis of cell populations withinhuman tumors than by conventional techniques. The microdissectiontechnique of the present invention may be used in combination with anumber of different technologies that allow for analysis of enzymes,MRNA and DNA from pure populations or subpopulations of particular celltypes. For example, DNA can be microdissected, using the techniques ofthe present invention, from normal epithelium, pre-malignant lesions,and invasive cancer in the same patient's single tissue sections. THisRNA can then be used to generate differential gene expression librariesby standard RT PCT. These libraries represent cellular stages of cancerprogression. As such, they can be used to screen for cancer diagnosticand prognostic markers.

This simple technique may have utility in characterizing proteasedistribution during human tumor invasion, precisely determining proteaseexpression in tumor and/or stromal cell populations as an indicator oftumor aggressiveness, and monitoring the effectiveness of anti-proteasetherapeutic agents in inhibiting protease activity at the tumor-stromalinterface. In addition, combination of this microdissection techniquewith PCR, RT PCR, differential display and SSCP may identify geneticalterations in specific subpopulations of tumor or stromal cell thatwould not be evident in heterogeneous human tumor samples.

The present invention has applications in routine diagnosis of humantumors including microdissection of pre-malignant lesions of all typesof cancer, genetic analysis of infectious diseases, gene therapy, tissuetransformation, and gene localization and analysis of transgenicanimals. Additional applications of this technique include analysis ofthe genotype, cellular products, or infesting organisms of rarepopulations such as Monocytes infected with drug resistant organisms,Reed-Stemberg cells of Hodgkins disease, Kaposi's sarcoma cells, stemcells, and vessel cells. Moreover, genetic analysis, or identificationof, micro-organisms infesting microscopically visualized cells intissues, lymph nodes or inflammatory areas can also be accomplished withhigh precision

Although the present invention has been described with reference toparticular means, materials and embodiments, from the foregoingdescription, one skilled in the art can easily ascertain the essentialcharacteristics of the present invention and various changes andmodifications may be made to adapt the various uses and characteristicswithout departing from the spirit and scope of the present invention asdescribed by the claims which follow.

What is claimed is:
 1. A transfer surface with an adhered extractedsample of material from a tissue sample wherein the adhered extractedsample is a cellular material extracted and collected for analysiscomprising: a selectively activatable transfer surface which only uponlocal activation at a selected region by electromagnetic radiation hascorrespondingly local adhesive characteristics to the material of thetissue sample; at least one locally activated selected region activatedby electromagnetic radiation to have correspondingly local adhesivecharacteristics to the material of the tissue sample upon the transfersurface; adhered extracted sample of material from a tissue sampleadhering to the at least one locally activated selected region; and,tissue sample absent from the transfer surface at regions which are notactivated by electromagnetic radiation.
 2. A transfer surface with anadhered extracted sample of material from a tissue sample according toclaim 1 comprising: a plurality of locally activated selected regionsupon the transfer surface; and, adhered extracted sample of materialadhering to the plurality of locally activated selected regions.
 3. Atransfer surface with an adhered extracted sample of material from atissue sample according to claim 1 where: the transfer surface isplanar.
 4. A transfer surface with an adhered extracted sample ofmaterial from a tissue sample according to claim 1 where: transfersurface is transparent.
 5. A transfer surface with an adhered extractedsample of material from a tissue sample according to claim 1 where: thetransfer surface includes a backing having an activatable coatingthereon.
 6. A transfer surface with an adhered extracted sample ofmaterial from a tissue sample according to claim 1 where: the transfersurface has been activated with a laser.